JPS6279781A - Production of peroxidase - Google Patents

Abstract

PURPOSE:To improve the productivity of peroxidase useful as a clinical diagnostic, by culturing a basidiomycete belonging a Coprinus genus and capable of producing peroxidase in a medium containing an iron salt. CONSTITUTION:A basidiomycete belonging to Coprinus genus, Coprinaceae family and capable of producing peroxidase [e.g. Coprinus macrorhizus K-1330 (FERM BP-648)] is cultured in a peroxidase-production medium containing an iron salt (e.g. sulfate, chloride, nitrate, etc.). The concentration of the iron salt in the medium is suitably 0.01-0.50wt/vol%, preferably 0.08-0.20wt/vol%.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for producing peroxidase.

More specifically, the present invention relates to a method for culturing basidiomycetes and producing peroxidase from the culture.

[Conventional technology]

Peroxidases are generally widespread in the plant kingdom.
Especially horseradish, figs, and two important ingredients:
Due to the fact that the peroxidase content in the roots of horseradish is the highest, peroxidase is industrially produced from horseradish and used in clinical diagnostic reagents, such as in combination with various oxidizing enzymes, in serum. It is widely used for quantifying glucose, total cholesterol, etc., or as a labeling enzyme in enzyme immunoassay.

Further, peroxidases of microbial origin include cytochrome C peroxidase and NADH peroxidase produced by bacteria and filamentous fungi, but these have different effects from peroxidases of plant origin such as horseradish and cannot be used as clinical diagnostic reagents.

Recently, the genus Alternaria, Cochliopolus, Pericyularia, and Carpularia (Japanese Patent Application Laid-open No. 57-99192)
It has been reported that microorganisms of the genus Bacillus (Japanese Patent Application Laid-Open No. 179488/1988) produce peroxidase that can be used as a clinical diagnostic reagent. However, these strains produce only a small amount of peroxidase, making them disadvantageous for industrial production.

The present inventors conducted intensive studies on peroxidase produced by Basidiomycetes as a method for industrially producing peroxidase, and as a result, found that Basidiomycetes belonging to the genus Cotypica have excellent properties as clinical diagnostic reagents in culture. discovered that peroxidase with
9-250900).

[Problem that the invention seeks to solve]

The present inventors have conducted intensive studies to further increase the production amount of peroxidase produced by the basidiomycetes belonging to the genus Cotypica, and as a result, we have added iron salts to the medium for culturing the basidiomycetes. The present inventors have discovered that the amount of peroxidase produced can be increased 2 to 4 times by culturing in the same medium without iron salts, and have completed the present invention.

Therefore, an object of the present invention is to provide a method for producing peroxidase, which has excellent properties as a clinical diagnostic reagent, at a lower cost in an industrial manner by culturing a basidiomycete belonging to the genus Cotylus.

[Means for solving problems]

To summarize the present invention, the present invention relates to a method for producing peroxidase, in which a basidiomycete belonging to the genus Cotypica and capable of producing peroxidase is cultured in a medium containing iron salts, and peroxidase is extracted from the culture. It consists of collecting.

The peroxidase obtained by the method of the present invention is peroxidized water fl
In the presence of
-Toluidine (hereinafter abbreviated as EHMT) and 3-methy/L/-2-benzothiazolinone hydrazone (hereinafter referred to as MB)
It can be used as a clinical diagnostic reagent because it develops color using dimethylaniline (abbreviated as TH) as a hydrogen donor.

The basidiomycetes used in the present invention include Coptaceae (Cop).
rinaceae) Coprinus
), such as Coprinus macrorhizas K.
-1330 (Coprinus macrorhiz
us (Japanese name: Nenaganohi toyota mushroom). This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, as FAIKEN Article No. 648.

To explain the method of the present invention in more detail, the nutrient source added to the striped area may be one that can be used by the strain used and can produce peroxidase, and the carbon source may include, for example, glucose, starch, sucrose, maltose, lactose, Glycerol, dextrin, fats and oils, etc. can be used, and suitable nitrogen sources include yeast extract, peptone, defatted soybean, corn steep liquor, and meat extract. In addition, inorganic and metal salts such as phosphates, potassium salts, and magnesium salts may be added, as well as vitamins and growth-promoting factors.

In order to increase the production of peroxidase, we conducted a more detailed study of the culture medium components, and found that the addition of various iron salts to the culture medium was the most effective way to increase peroxidase production. Examples of iron salts added to the medium every day include iron sulfate, iron chloride, iron nitrate, etc., and these can be used alone or in a mixture of two or more.

In addition, the amount of iron salt added to the medium is 0.01% ~
0.50%, preferably 0.108% to 0.20% is suitable. Note that the amount of iron salt added is expressed in weight/volume %.

When culturing basidiomycetes, the amount of peroxidase produced varies greatly depending on the culture conditions, but generally the culture temperature is 20 to 35°C, the pH of the medium is 4 to 8, and peroxidase production can be reduced by aeration and agitation culture for 4 to 12 days. reach the highest. It goes without saying that culture conditions should be set to maximize peroxidase production depending on the bacterial strain used, medium composition, etc. The peroxidase produced by the strain of the present invention is mainly present in the culture F solution, and can be prepared by adding 20-80 w/v% of a precipitating agent such as ammonium sulfate to the culture P solution, or by adding 50-80 v% of an organic solvent such as alcohol, acetone, etc. /v%, it is separated as a precipitate. The obtained precipitate is desalted by ultrafiltration, dialysis or Sephadex treatment to obtain a crude enzyme solution. To purify the obtained crude enzyme solution, 0.0
DEAE buffered with 1M phosphate buffer (pH 7,0)
- Adsorb the crude enzyme solution on a column of Sepharose (CL-6B), and remove the adsorbed material with 0.02M phosphate buffer (pH 7,0
) After washing, add 0.1 M IJ acid buffer (p) 17.
0) and collect the active fraction. Next, this active fraction was concentrated and desalted by ultrafiltration, and then 0.01M phosphate buffer (
DEAE-Sepharose (C
L-6B) was again adsorbed on the column, and the adsorbed material was reduced to 0.02
After washing with M phosphate buffer (pH 7,0), 0.05
The active fraction is collected by elution with M phosphate buffer (pH 7.0).

This active fraction is dialyzed against distilled water and then freeze-dried to obtain 2-purified enzyme powder. This enzyme powder is single in polyacrylamide gel disk electrophoresis.

The enzymatic and physicochemical properties of the peroxidase of the present invention are as follows.

(1) Action: This enzyme acts very specifically on hydrogen peroxide, and in the presence of hydrogen peroxide, it mediates the oxidation of various compounds that can serve as hydrogen donors. Its mechanism of action is as shown below.

H20□+AH2→2H20+ A Note that AH2 represents a hydrogen donor, and A represents an oxidized hydrogen donor.

(2) Specificity for hydrogen donors: When 4-AA and various compounds are used in combination as hydrogen donors, the hydrogen donors of this enzyme are as shown in Table 1 Phenol 500 100 Resorcinol 10 Pyrocatechol " 58 Hydroquinone 10 α- Naphthol " 182.4-dichloroph 520 4phenol 2.4-dichloroph " 115 enol 2.6-cycloph " 154 enol 2.4.6-)rif " 960 lophenol dimethylaniline 550 21 diethylaniline " 49EHMT
62 Also, MBT
H and dimethylaniline, diethylaniline or EH
We investigated the specificity when combining MTs as hydrogen donors (second main), each t
“η し １ い JA ＾ −−−＋ I
When +1 is used as a hydrogen donor, the relative value is 10
It was set to 0.

Dimethylaniline 590 50Diethylaniline “ 49EHMT
41 (4-AA-
Phenol 500 100) (3) Optimal pH and pH stability: The optimal pH of this enzyme is as shown in the graph in Figure 1 (
It has high activity around pH 7.0. pH 3-3.
5 is acetate buffer, pH 6-85 is phosphate buffer, pH 9
~9.5 used borate buffer. Figure 2 shows the pH stability when this enzyme was treated at 37°C for 60 minutes at each pH. As is clear from FIG. 2, this enzyme is stable between pH 6.0 and pH 7.5.

(4) Optimal temperature and thermostability: The optimal temperature of this enzyme is as shown in the graph of Figure 3.
It has a temperature of around 35°C to 40°C. Figure 4 shows the thermal stability of this enzyme when it was treated at pH 7.0 and at various temperatures for 10 minutes. This enzyme was stable up to 45°C.

(5) Molecular weight: The molecular weight of this enzyme is approximately 37,000 when measured by Ge)v filtration using Sephacryl S-200 (manufactured by Pharmacia);
Approximately 41 by 5DS-polyacrylamide gel electrophoresis
(6) Uniformity: Disk electrophoresis was performed using 7.5% polyacrylamide gel (p149.4), and protein staining revealed one single stained band. Also, 5DS
-Polyacrylamide gel electrophoresis also showed a single band.

(7) Isoelectric point: The isoelectric point of this enzyme determined by focusing electrophoresis using Pharmalite (pH 3-10, manufactured by Pharmacia) is pL
:3.4 to 3.5.

(8) Effects of inhibitors, metal ions, and metal chelating agents: This enzyme has 88 generations of potassium cyanide, sodium azide,
It was inhibited by thiourea and the like (Table 3).

No additives 100 Iodochoic acid 100L-cysteine 105 EDTA 97 Dithiothreitol 95 CuS0. 1
05 Thiourea 57 MnC1,86
Potassium cycinide 22 FeC1107 Sodium azide 52 BaC1□ 96
PCMB' 98 CoC1□ 98F
MS-rice 98 ZnC1z 85 Sodium fluoride 103 5nC1□ 92 Sodium sulfide 82 NiSO492α, α'
-Dipyridyl 103 HgCl26 US phenylmethylsulfonyl fluoride (9) visible region absorption spectrum; This spectrum is shown in FIG.

α0) Enzyme activity measurement method: Peroxidase activity was measured using 4-AA-phenol, which is used as a hydrogen donor in clinical diagnostic reagents. That is, 5mM hydrogen peroxide solution 0.1mJ10.
3M IJ acid buffer (pH 7,0) 1.0d, 24
．． 6mM 4-AA solution 0.1 ml, 0.42M phenol solution Q, 1 ml, water 1.6-ml and appropriately diluted enzyme solution 0.1 ml, reaction solution volume 3, 0 ml 37
After reacting at ℃ for 60 seconds, the absorbance at 500 nm (OD sample/L/) is measured. Separately, as a control, 0.11% of water was added instead of the hydrogen peroxide solution, and the absorbance (OD blank) was measured in the same manner as ΔOD. . (OD sample-〇D blank) was determined. Peroxidase activity is determined by the following formula.

6.17XtXVs = △OD5°. X4.86 Although illustrated with examples, the present invention is not limited to the following examples.

Example 1 Effect of addition of iron salts on peroxidase production Glucose 2%, Ebios 0.5% and Agar 1.5%
Coprinus macrorhizas -1330 (Feikoken Joyori No. 648) was inoculated into a slanted medium having the composition of (Ebios medium), and cultured for one week at 25°C to prepare a seed culture. Glucose 2%, yeast extract 0.3%, peptone 1%, KH
100ml of a medium with a composition of 2PO40.3%, Mg5O,7H,0.1% was dispensed into a 500M Erlenmeyer flask, and FeSO4.7H20 was added 0%, 0.04%, 0.
．． 08%, 0.12%, 0.16%, 0.20%, 0.
They were added to each flask to give a concentration of 24% and 0.32%, sterilized at 120°C for 20 minutes, cooled, and inoculated with the above-mentioned inoculum, and incubated at 26°C for 5 to 10 minutes.
The cells were cultured with shaking at 100 rpm per minute for days, and samples were taken over time. Filter these culture fluids to remove bacterial cells,
Table 4 shows the peroxidase activity (maximum activity) of each of the obtained p solutions.

No additives 19.4 0.04 28.0 0.08 46.40.12
75.50.16
80.20.20 6
6.00.24 36.80
．． 32 17.0 As is clear from Table 4, when iron salts were added to the medium, the production amount of peroxidase was significantly increased. In addition, Fe50.・7
The optimum amount of H20 added was 0.08 to 0.20%.

Example 2 Coprinus macrorhizas cultured in the Ebios medium of Example 1 was treated with -1330 (Feikoken Joyori No. 648) with 2% glucose, 0.3% yeast extract, 1% peptone, and KH.
, PO40,3%, MgSO4.7H,OO,1% and FeSO4.7H200,16%.
m/ and sterilized (120℃, 20 minutes) 50
It was inoculated into a 0 m Erlenmeyer flask, and cultured with shaking at 100 rpm per minute at 27°C for 10 days. After the culture was completed, the bacterial cells were removed by filtration to obtain liquid F. The peroxidase activity was 79.4 units/ml.

Example 3 Coprinus macrorhizas cultured in the Ebios medium of Example 1 was treated with -1330 (Feikoken Joyori No. 648) with 2% glucose, 0.3% yeast extract, 1% peptone, and KH.
2PO40,3% and MgSO4'7H20,0,1
Dispense 100 rLl of % medium and sterilize it (120°012
0 minutes) into a 500 mJ Erlenmeyer flask,
The cells were cultured at 26°C for 7 days and used as a seed culture. Glucose 2%, yeast extract 0.3%, peptone 1%, KH2PO
40.3%, MgSO4Φ7H200.1%, FeSO
4.7H200, 16% and antifoaming agent (C manufactured by NOF Corporation)
B-442) Culture medium 201 with a composition of 0.02% to 301
The mixture was placed in a large jar fermenter and sterilized at 120°C for 20 minutes. After cooling, inoculate 100 d of the above seed culture solution, and
The cells were cultured at 6°C for 8 days under conditions of an aeration rate of 131 rpm and a stirring rate of 270 revolutions per minute. After the culture was completed, the bacterial cells were removed by filtration to obtain liquid F.

The peroxidase activity was 84.1 units/TILl. Add 80 g of ammonium sulfate to 17 J of this culture solution.
% saturation, and after standing for day and night, the obtained ammonium sulfate precipitate was added to about 500% of 0.01M phosphate buffer (pH 7,
0). This crude enzyme solution was heated in an ultra-furnace vortex to a DEAE-Sepharose (CL-6B) column (diameter 5.0 crrL x After washing the adsorbed material with 0.02 MIJ phosphate buffer (pH 7,0), it was eluted with 0.1 M phosphate buffer (pH 7,0) and the active fraction was collected. This active fraction is then concentrated by ultrafiltration,
After desalting, the adsorbed material was adsorbed again onto a DEAE-Sepharose (CL-6B) column (diameter 2.5 crrL x length 4 crrL) buffered with 0.01M phosphate buffer (pH 7.0), and the adsorbed material was absorbed at 0.01M phosphate buffer (pH 7.0). 02M phosphate buffer (p) 17.0
) and then eluted with 0.05M phosphate buffer (pH 7.0) to collect the active fraction. This active fraction was dialyzed against distilled water and then freeze-dried to obtain purified enzyme powder 1007Tn9. The specific activity of this powder was 1182 units/da. This enzyme powder was uniform in polyacrylamide gel disk electrophoresis. Semen No. 5 culture p solution of above 240000 1430000 596
100 Ammonium sulfate salting out 40540 1427000
35.2 99.8 Ultrafiltration 16390 14
11000 86.1 98.7 Reference Example 1 -1330 (Feikoken Joyori No. 648) was added to Coprinus macrorhizas cultured in the Ebios medium of Example 1 with 2% glucose, 7 yeast extract, 0.3%, Peptone 1%, K
H2PO40.3% and MgSO4.7H200.1
Dispense 100% of the medium and sterilize it (120℃, 2
The cells were inoculated into a 500 ml Erlenmeyer flask and cultured at 27°C for 10 days with shaking at 100 rpm per minute. After the culture was completed, the bacterial cells were removed by filtration to obtain a p solution. This peroxidase activity was 20.0 units/4.

Reference Example 2 Coprinus macrorhizas cultured in the Ebios medium of Example 1 was treated with -1330 (Feikoken Jokyo No. 648) with 2% glucose, 13% yeast extract O, 1% peptone, and KH.
2PO40,3% and MgSO4-7H200,1%
100 ml of the culture medium was dispensed and inoculated into a sterilized (120°C, 20 minutes) 500 ml Erlenmeyer flask, and cultured at 26°C for 7 days to obtain a seed culture solution. Glucose 2%, yeast extract 0.3%, peptone 1%, KH2PO40.3
%, MgSO4・7H200,1%, CuSO4・5H
200,005% and antifoaming agent (CB-4 manufactured by NOF Corporation)
42) Medium 201 with a composition of 0.02% was placed in a 301 volume jar fermentor and sterilized at 120°C for 20 minutes. After cooling, 100 d of the above seed culture solution was inoculated and cultured at 26° C. for 6 days under conditions of an aeration rate of 13 J/min and a stirring rate of 270 revolutions/min. After the culture was completed, the bacterial cells were removed by filtration to obtain an R liquid.

The peroxidase activity was 41.5 units/ml. Add 80% ammonium sulfate to this culture OF solution 171.
Add the ammonium sulfate precipitate so as to saturate it and - After standing for a day and night, add about 500% of the obtained ammonium sulfate precipitate to 0.00%. OIM phosphate buffer (pH 7
,0). Concentrate this crude enzyme solution by ultrafiltration,
After desalting, the adsorbed material was adsorbed onto a DEAE-Sepharose (CL-6B) column (diameter 5.0 cm x length 4α) that had been loosened in advance with 0.01H phosphate buffer (pH 7.0). After washing with 0.02M phosphate buffer (pH 7,0), the active fraction was collected by elution with 0.1M phosphate buffer (pH 7,0), and the active fraction was concentrated by ultrafiltration in nine people. After desalting, a column of DEAE-Sepharose (CL-6B) buffered with 0.01M phosphate buffer (pH 7.0)
The adsorbed substance was adsorbed again to 2.5 C1rL x 4crIL in diameter, washed with 0.02M phosphate buffer (pH 7.0), and eluted with 0.05M phosphate buffer (pH 7.0) to determine the activity. Collected classifications. This active fraction was dialyzed against distilled water and then lyophilized to obtain purified enzyme powder 494rn9.

The specific activity of this powder was 1180 units/insert.

Is this enzyme powder a poly7 acrylamide gel disc? le
Table 6 shows the tsun mother T degree of Bt-Chikit and Heji Jμ in terms of method @I+.

Culture r solution 237500 707000 2.98
100 Ammonium sulfate salting out 41200 724000
17.6 102 limit F' over 17000 7030
00 41.4 99.4 [Effects of the Invention] According to the present invention, the production amount of peroxidase useful as a clinical diagnostic reagent is increased by 2 to 4 times compared to the conventional method, and a manufacturing method suitable for advantageous industrial production has been established. offered.

[Brief explanation of drawings]

Figure 1 shows the pH of peroxidase obtained by the present invention.
represents the relationship between and activity. Figure 2 shows three enzymes according to the present invention.
p after treatment for 60 min at each pH at 7°C.
Figure 3 shows the relationship between H and activity, Figure 3 shows the relationship between temperature and activity, and Figure 4 shows the relationship between temperature and activity after 10 minutes of treatment at each temperature at pH 7 and 0. Fifth
The figure shows the visible region absorption spectrum of the enzyme according to the present invention (enzyme concentration 0.057%, pH 7.0).

Claims (1)

[Scope of Claims] 1. A method for producing peroxidase by culturing peroxidase-producing bacteria belonging to the genus Cotylus in a medium and collecting peroxidase from the culture, characterized in that the medium contains an iron salt. manufacturing method. 2. The method for producing peroxidase according to claim 1, wherein the iron salt is one or a mixture of two or more of iron sulfate, iron chloride, and iron nitrate.